The objective of this research is to elucidate the control mechanisms involved in the regulation of de novo pyrimidine bio-synthesis in mammalian cells. The first three enzymes of the pathway; carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase, are associated with a single polypeptide chain. This multifunctional protein, complex A, also exhibits allosteric transitions. The protein, isolated from a mutant SV40 transformed hamster cell line, will be cleaved by controlled proteolysis to determine the number, function and arrangement of autonomously folded structural domains. These studies will employ chemical modification, peptide mapping, end group analysis and crosslinking experiments. The structure and interconversion of the oligomeric forms of the complex will be studied by gel filtration and ultracentrifugation. Studies will focus on the unique functional properties such as channeling, reduction in transient time, coordinate effects and catalytic facilitation and the interrelationship of these properties to the allosteric control mechanisms. The kinetics of the system will be simulated by a computer model, to assess the extent to which compartmentation of intermediates can account for the functional properties of the complex. Using a combination of techniques, enzymatic assays, immunoprecipitation, gel electrophoresis and fluorescent microscopy, the activity, oligomeric form, intracellular concentration and location and the turnover of the molecule will be studied in cultured cells. For these studies normal and viral transformed cells and cells in different growth phases or in different phases in the cell cycle will be compared. While we expect the control mechanisms to operate normally in these cells, they differ widely in pyrimidine biosynthetic activity and will thus be useful in identifying signals which trigger alterations in the pathway and deciphering which controls observed in vitro are operative in the cell.